Quantitation of fiber growth in transplanted central monoamine neurons

Summary Nerve fiber production by central noradrenaline (NA), dopamine (DA), and 5-hydroxytryptamine (5-HT) neurons was studied using immature brain tissue containing locus coeruleus, substantia nigra, or ventro-caudal medulla oblongata respectively, homologously grafted to the anterior chambers of rat eyes. A method was developed for quantitation of the fiber growth that occurs on the sympathetically denervated host irides as observed in whole mounts using Falck-Hillarp fluorescence histochemistry and by the uptake of ³H-metaraminol into the irides. Survival and growth in oculo of the three different areas were characterized by direct observations through the cornea in vivo for a number of pre- and postnatal stages of development of the donors, and the findings correlated to the degree of monoamine nerve fiber production on the host irides. The growth of fetal locus coeruleus transplants on irides was quantified using both fluorescence microscopical measurements of innervated areas and uptake of ³H-metaraminol. The uptake was well correlated to the histochemical measurements on individual irides, thus validating the fluorescence microscopical measurements of fiber production. The fiber growth of fetal locus coeruleus grafts on irides was followed for 20 weeks. The nerves increased in number and uptake capacity approximately linearly for 6 weeks whereafter the increase rapidly levelled off. On average, the final amount of nerve production by fetal locus grafts did not cover more than 1/3 of the host iris surface, and the average uptake of ³H-metaraminol by these nerves did not exceed 60% of that found in sympathetically intact control irides. The locus grafts produced a similar amount of fluorescent fibers in the host iris independent of the crown-rump length stage of the donor fetus and the final size of the transplants in oculo.The survival and growth of NA, DA and 5-HT neurons grafted from various postnatal donor rats was also followed by fluorescence microscopy. Locus coeruleus grafts produced markedly more fibers than the two other types of grafts when the donor was one week old or less, and DA grafts produced the least fibers of the three. Even from one month old donors some MA neurons survived grafting. Also, using prenatal donars, the locus coeruleus grafts produced many more fibers on the irides than did the DA grafts. It was concluded that the intraocular transplantation technique is very suitable for quantitative studies of nerve fiber production by immature monoamine neurons, and that it should be possible to study many other neuron systems in similar ways with this technique.Supported by the Swedish Medical Research Council (04X-03185), Magnus Bergvalls Stiftelse and Karolinska Institutets Fonder. The skilful technical assistance of Miss Ingrid Strömberg, Miss Maud Eriksson and Miss Gerd Boëtius is gratefully acknowledged. Thanks are due to Swedish Pfizer for the generous supply of Nialamid^®     

The innervation of the salivary gland of the moth,Manduca sexta

Summary The salivary glands of the moth, Manduca sexta (Insecta: Sphingidae), are unlike most other salivary glands in that they are innervated from one source only. Vital staining of nerves with methylene-blue reveals numerous fine nerves extending to the glands from the oesophageal nerve, a part of the stomatogastric or visceral nervous system. Light and electron microscopy confirm that only the fluid-secreting cells, confined to a discrete region in these glands, are innervated. Axons with or without glial wrappings are found in intercellular spaces between fluid-secreting cells. Axons lacking a glial sheath contain, after glutaraldehyde-osmium tetroxide fixation, large granular and small agranular vesicles. In nerve endings in glands fixed with permanganate these smaller vesicles are granular, having the electron-dense cores characteristic of monoamine-containing neurons. These nerve endings with synaptoid areas are in close (direct) contact with the fluid-secreting cells.I am grateful to Professor T. Weis-Fogh for accommodation in the department of Zoology and to Dr. Nancy Lane for use of A.R.C. facilities and advice. Thanks are also due to Drs. M. J. Berridge, S.H.P. Maddrell, and W. T. Prince and Mr. R. A. Leslie for helpful discussion. Financial assistance from Clare College, Cambridge is gratefully acknowledged.     

Genetic organization of the E. coli chromosome around the structural gene for initiation factor IF3 (infC).

Summary A set of transducing phages carrying varying lengths of the E. coli chromosome around the structural gene for initiation factor IF3 (infC) was derived from p2 which is known to cary, besides infC, the structural genes for the subunit of phenylalanyl-tRNA synthetase (pheS), the subunit of phenylalanyl-tRNA synthetase (phetT) and the structural gene for threonyl-tRNA synthetase (thrS). The E. coli coding content of these derived phages was analysed by genetic complementation of a set of mutants and by SDS-polyacrylamide gel analysis of the proteins synthesized in UV irradiated cells infected with these phages. The segregation pattern of the different genes among these derived phages indicates that the order of the genes is pheT-pheS-P12-(infC, thrS) where infC is probably between P12 and thrS. P12 is the structural gene of a 12,000 molecular weight unidentified protein.Abbreviations PRS (EC 6.1.1.20) phenylalanyl-tRNA synthetase - TRS (EC 6.1.1.3) threonyl-tRNA synthetase - IF3 Initiation factor IF3 - SDS Sodium dodecyl sulfate - PP^R pyrophosphate resistant - PP^S pyrophosphate sensitive     

Brain tissue transplanted to the anterior chamber of the eye: 2. Fluorescence histochemistry of immature catecholamine and 5-hydroxytryptamine neurons innervating the rat vas deferens

Summary Small pieces of the wall of the rat vas deferens were homologously transplanted to the anterior chamber of the eye together with small pieces of embryonic brain stem containing either developing noradrenaline (NA) cells of the locus coeruleus or 5-hydroxytryptamine (5-HT) neurons of the developing raphe system. The eyes of the recipients were sympathetically denervated. The double transplants became rapidly vascularized from the host iris. After 31/2 months the irides, together with their two transplants were analyzed by Falck-Hillarp fluorescence microscopy. Both the NA and the 5-HT neurons had survived and matured in the eye. Fluorescent varicose nerve terminals of the NA and 5-HT type respectively were found in all three potential receptor areas, i.e. within the CNS transplants, in the host irides and in the vas deferens transplants. In the latter, the newly formed monoamine nerve terminals arborized mainly within a well developed smooth muscle layer. The density of such new fibres was higher than or similar to that of the normally present sympathetic plexus in areas of the transplant close to the CNS transplant and lower in areas at a distance from the CNS transplant. It is concluded that immature central NA and 5-HT fibres are able to grow simultaneously into different types of sympathetically denervated smooth muscle tissues to form networks of fibres in the receptor organs resembling the normal sympathetic innervation.Supported by the Swedish Medical Research Council (04X-3185), and by grants from Magnus Bergvalls Stiftelse, Tidningen Expressens Prenatalforskningsfond and Karolinska Institutets fonder. We thank miss Ingrid Strömberg for skilful technical assistance. Nialamide^® was generously donated by Swedish Pfizer AB.     

Recombinant Components of the EcoRII Restriction–Modification System: Restriction Endonuclease Can Interact with DNA · RNA Duplexes

To obtain recombinant restriction endonuclease (R) and methylase (M) of the EcoRII restriction–modification system, bacterial strains overproducing their functional hexahistidine derivatives were constructed. Active full-length R·EcoRII was produced only in cells that also expressed M·EcoRII from a multicopy plasmid. Recombinant R·EcoRII bound with hybrid DNA·RNA duplexes.     

Effects of stimulating midbrain central gray matter on neuronal response in the caudal trigeminal nucleus to peripheral nerve stimulation

Stimulating the midbrain central gray matter (CGM) with trains of 10–20 stimuli was found to inhibit response to electrical stimulation of infraorbital nerve and tooth pulp A-alpha and A-delta afferents at 100 msec intervals in 65% of the caudal trigeminal nucleus in neurons tested during experiments on cats under chloralose-Nembutal anesthesia. Response was inhibited most effectively in convergent neurons (activated by stimulating infraorbital nerve and tooth pulp A-alpha and A-delta afferents) to stimulating tooth pulp (0.76) and, to a somewhat lesser degree, to stimulation of A-alpha afferents (0.6). For high-threshold neurons (activated by stimulating infraorbital nerve and tooth pulp A-delta afferents), success rate of inhibiting response under the effects of CGM stimulating measured 0.71 and 0.48 for low-threshold cells (activated by stimulating infraorbital nerve A-alpha afferents). Stimulating CGM produced an excitatory response in 10 caudal trigeminal nucleus neurons within 7.5–20 msec; after this neurons showed no reaction to peripheral nerve stimulation for a 200–450 msec period. The possible involvement of these neurons in pressing the mouth-opening reflex produced by CGM stimulation is discussed in this article.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 20, No. 6, pp. 729–736, November-December, 1988.     

Fluorescence-microscopical demonstration of a population of gastro-intestinal nerve fibres with a selective affinity for Quinacrine

Summary A population of nerve fibres in the gastro-intestinal tract of mice showing a high affinity for quinacrine was revealed by fluorescence microscopy. Similar results were obtained in rats and guinea pigs. Whole-mounts of sheets of the smooth muscle layer following incubation in 10^-6-10^-7 M quinacrine for 15–60 min revealed fine fluorescent varicose nerve fibers in the myenteric plexus of Auerbach both around nerve cell bodies and in the interconnecting strands. Many fibers were also present between the strands of the plexus, especially running parallel to the circular muscle layer. Such fibers were not seen in similarly quinacrine-incubated irides. A proportion of the cell bodies in Auerbach's plexus also showed quinacrine accumulation. These cells were apparently smaller neurons, sometimes with fluorescent processes. Intraperitoneal injections of quinacrine failed to demonstrate nerve fibers, but some cell bodies in Auerbach's plexus were positive. Subsequent paraformaldehyde treatment for monoamine visualization showed persistent adrenergic nerve terminals in the intestine and iris. These nerves seemed to be fewer and had a more yellow fluorescence than normally. The identity of the quinacrine-positive fibers is discussed with respect to recent suggestions that purinergic, substance P, enkephalin, and somatosin-containing nerves, in addition to adrenergic and cholinergic nerves, are present in the gut wall.Supported by the Swedish Medical Research Council (04X-03185). Magnus Bergvalls Stiftelse and Karolinska Institutets Fonder. For generous gifts of Mepacrine we thank Winthrop, Skärholmen, Stockholm, Sweden. The skilful technical assistance of Miss Gerd Boetius and Miss Maud Eriksson is gratefully acknowledged     

Orthopaedic implant related metal toxicity in terms of human lymphocyte reactivity to metal‐protein complexes produced from cobalt‐base and titanium‐base implant alloy degradation

Metal toxicity from sources such as orthopaedic implants was investigated in terms of immune system hyper-reactivity to metal implant alloy degradation products. Lymphocyte response to serum protein complexed with metal from implant alloy degradation was investigated in this in vitro study using primary human lymphocytes from healthy volunteers (n = 10). Cobalt chromium molybdenum alloy (CoCrMo, ASTM F75) and titanium alloy (Ti6Al4V, ASTM F136) beads (70 m) were incubated in agitated human serum at 37 degrees Celsius to simulate naturally occurring metal implant alloy degradation processes. Particulate free serum samples, which were incubated with metal, were then separated into molecular weight based fractions. The amounts of soluble Cr and Ti within each serum fraction were measured and correlated with lymphocyte proliferation response to the individual serum fractions. Lymphocytes from each subject were cultured with 11 autologous molecular weight based serum fractions either with or without added metal. Two molecular weight ranges of human serum proteins were associated with the binding of Cr and Ti from CoCrMo and Ti implant alloy degradation (at < 30 and 180–330 kDa). High molecular weight serum proteins ( 180 kDa) demonstrated greater lymphocyte reactivity when complexed with metal released from CoCrMo alloy and Ti alloy than with low (5–30 kDa) and midrange (30–77 kDa) serum proteins. When the amount of lymphocyte stimulation was normalized to both the moles of metal and the moles of protein within each fraction (MetalProtein Complex Reactivity Index, MPCRI), Cr from CoCrMo alloy degradation demonstrated approximately 10 fold greater reactivity than Ti in the higher molecular weight serum proteins ( 180–250 kDa). This in vitro study demonstrated a lymphocyte proliferative response to both CoCrMo and Ti alloy metalloprotein degradation products. This response was greatest when the metals were complexed with high molecular weight proteins, and with metalprotein complexes formed from CoCrMo alloy degradation.     

Brain tissue transplanted to the anterior chamber of the eye

Summary Small pieces of fetal rat brain selected to contain a high number of noradrenaline (NA), dopamine (DA), or 5-hydroxytryptamine (5-HT) neuroblasts were transplanted to the anterior chamber of the eye of adult rats. The sympathetic ground plexus of the host iris was removed by superior cervical ganglionectomy so that transmitter mechanisms of the different central monoamine fibers innervating the iris could be selectively studied after intraocular maturation. Such irides, containing NA, DA, or 5-HT nerve terminals were incubated with radiolabelled transmitters and then stimulated by an electrical field while superfused, to investigate the spontaneous and stimulation-induced release of amine, both in drug-free buffer and buffer containing drugs acting on monoamine receptors.The central monoamine neurons of all three types were able to take up exogenous amines and release them upon stimulation by an electrical field, in much the same way as corresponding nerves in situ in slices of cerebral cortex (NA, 5-HT) or olfactory tubercle (DA).The -adrenergic receptor blocking agent phentolamine increased the stimulation-induced release of ³H-NA from central NA fibers on the iris significantly. The dopamine receptor stimulating agent apomorphine decreased the stimulation-induced release of ³H-DA from central DA fibers on the iris. Pimozide, a DA receptor blocking drug tended to increase the ³H-DA release. The 5-HT receptor stimulating agent ergocornine tended to reduce the stimulation-induced release of ³H-5-HT from central 5-HT fibers on the iris. It was concluded that all three types of central monoamine nerve fibers develop essentially normal transmitter storage and release mechanisms also in an environment completely devoid of normal postsynaptic receptors. The drug experiments add strong support to the view that there are presynaptic monoamine receptors (autoreceptors) able to modulate transmitter release present on the monoamine nerve terminals.Supported by the Swedish Medical Research Council (04X-3185 and 04X-2330) and by grants from Magnus Bergvalls Stiftelse and Karolinska Institutets Fonder, we thank Miss Ingrid Strömberg and Miss Ulla Enberg for skilful technical assistance.     

Organization of the nervous system of physonectid siphonophores

Summary An antiserum to the sequence Arg-Phe-amide (RFamide) was used to stain the nervous systems of various physonectid siphonophores. In the stem of Nanomia bijuga, this antiserum stained an ectodermal nerve net, which was interrupted, at regular intervals, by transverse collars of neurons. Injection of Lucifer yellow into the giant axon of the stem showed that this axon was dye-coupled to an ectodermal nerve net that resembled the RFamide-positive network. Ectodermal nets of neurons were also found in the pneumatophore, gastrozooids, tentacles and tentilla. At the junctions of the pneumatophore, the gastrozooids, the dactylozooids and the gonozooids with the stem, and at the junctions of tentacles and tentilla, collars or rings of neurons occurred. The stem was connected to the phyllozooids and nectophores by muscular lamellae, which were bordered by chains of neurons. At the margin of the nectophores, an immunoreactive nerve ring was found. Connected to this ring and located in theseitliche Zapfen (sidely-located patche), were two agglomerations of nerve cells. On the upper side of the bell margin, positioned at 90° relative to the seitliche Zapfen, a delta-shaped neuronal structure was found. This structure was connected to the nerve ring and was associated with a muscle, which ran a short distance along the exumbrellar surface.The nervous systems of Agalma elegans, Forskalia edwardsi, Forskalia leuckarti and Halistemma rubrum resembled that of Nanomia bijuga in all major respects.     

Some parasympathetic neurons in the guinea-pig heart express aspects of the catecholaminergic phenotype in vivo

Summary In a histochemical study of intrinsic cardiac ganglia of the guinea-pig in whole-mount preparations, it was found that some 70–80% of the neurons express aspects of the catecholaminergic phenotype. These neurons have an uptake mechanism for L-DOPA, and contain the enzymes for converting L-DOPA, (but not D-DOPA) to dopamine and noradrenaline, i.e. aromatic L-aminoacid decarboxylase and dopamine -hydroxylase. Monoamine oxidase is also present within some of the neurons. In these respects, the neurons resemble noradrenergic neurons of sympathetic ganglia, so we refer to them as intrinsic cardiac amine-handling neurons. However, these neurons do not contain tyrosine hydroxylase and show little or no histochemically detectable uptake of -methyldopa, dopamine or noradrenaline, even after depletion of endogenous stores of amines by pre-treatment with reserpine. Noradrenergic fibres from the sympathetic chain form pericellular baskets around nerve cell bodies. The uptake of L-DOPA into nerve cell bodies is not prevented by treatment with 6-hydroxydopamine sufficient to cause transmitter-depletion or degeneration of the extrinsic noradrenergic fibres. Such degeneration experiments suggest that axons of the amine-handling neurons project to cardiac muscle, blood vessels and other intrinsic neurons. The cardiac neurons do not show any immunohistochemically detectable serotonergic characteristics; there is no evidence for uptake of the precursors L-tryptophan and 5-hydroxytryptophan or 5-HT itself, whereas the extrinsic noradrer ergic nerve fibres within the ganglia can take up 5-HT when it is applied in high concentrations.Abbreviations AChE acetylcholinesterase - DBH-IR dopamine -hydroxylase-like immunoreactivity - L-DOPA L-dihydroxyphenylalamine - 5-HT-IR 5-hydroxytryptamine-like immunoreactivity - 6-OHDA 6-hydroxydopamine - methyldopa L--methyl-dihydroxyphenylalanine - MAO monoamine oxidase - NPY neuropeptide Y - SIF small intensely fluorescent cells - TH-IR tyrosine hydroxylase-like immunoreactivity - VIP vasoactive intestinal polypeptide     

Chitin and β(1–3) glucan synthases in the protoplastic entomophthorales

(1–3) glucan and chitin synthases were studied in spontaneously produced protoplasts and in the mycelium (hyphal body) of the entomopathogenic Entomophthorale species Entomophaga aulicae, Conidiobolus obscurus and Entomophthora muscae. The absence of wall in protoplasts was correlated to an absence of chitin synthase and to a very low (1–3) glucan synthase activity, whereas these two polysaccharide synthases were present and active in the walled hyphal bodies. Physicochemical properties of chitin and (1–3) glucan synthases such as localization, optimum pH and temperature, activation by disaccharides and proteases were similar to those found in other fungi unable to spontaneously produce protoplasts and could not be related to the ability for protoplastic Entomophthorale species to produce and proliferate under a protoplast form. The absence or the low chitin and glucan synthase activites in Entomophthorale protoplasts was not due to an absence of proteolytic activation of the enzyme. However, all protoplast fractions contained inhibitory substances of glucan and chitin synthase activities. These inhibitors were stable and specific of the protoplast stage. They were not glucanase nor chitinase. These results suggest that the absence of wall synthesis in Entomophthorale protoplasts is due to a continuous inhibition of (1–3) glucan and chitin synthase activities by intracellular compounds and also for glucan synthase by protoplast medium constituents such as NaCl and fetal calf serum.Abbreviations BSA bovine serum albumin - DFP diisopropylfluorophosphate - EDTA ethylenediamine tetraaoetic acid - FCS fetal calf serum - GlcNAc N-acetylglucosamine - TCA trichloroacetic acid - 2 k pellet 2,000 g wall fraction - 140 k pellet 140,000 g particulate fraction - 140 k supernatant 140,000 g soluble fraction     

Expression of the β-subunit of β-conglycinin in seeds of transgenic plants

Soybean (Glycine max (L.) Merr.) seeds contain the storage protein -conglycinin, encoded by a multigene family. -Conglycinin consists of three subunits; , , and . A genomic clone for a -subunit of -conglycinin has been characterized by restriction-enzyme mapping and hybrid selected in-vitro translation followed by immunoprecipitation. In order to determine the developmental regulation of this -subunit gene, its expression was studied in seeds of transgenic petunia (Petunia hybrida) and tobacco (Nicotiana tabacum L.) plants. The -subunit expressed in seeds of petunia and tobacco was recognized by anti--conglycinin serum at a relative molecular mass of 53 000, equivalent to that of the native protein. Separation of the petunia-seed proteins by isoelectric focusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis showed that multiple isoelectric forms of the -subunit were produced. There was approximately a twofold variation in the accumulation of the -subunit protein in the mature seeds of transgenic petunia plants, each containing a single -subunit gene. However, the level of protein accumulation in mature seeds and the amount of -subunit mRNA in developing seeds was not correlated. Accumulation of the -subunit protein in transgenic seeds was less than the -subunit protein that accumulated in transgenic petunia seeds containing a single -subunit gene and less than the amount of the -subunit in mature soybean seeds which contain 8–13 -subunit genes. In transgenic tobacco plants, the accumulation of the -subunit protein in seeds was generally well correlated with the number of genes that were incorporated in the different transformants.Abbreviations kb kilobase - kDa kilodalton - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Intranuclear inclusions in pericytes of the hypothalamus of the rat

Summary This paper deals with the ultrastructure of two types of intranuclear inclusions, nuclear bodies and membranous lamellar bodies, present in hypothalamic pericytes of intact adult rats. The nuclear bodies exhibited simple and granular forms, whereas the membranous lamellar bodies were entirely made up of myelin-like membrane whorls.The occurrence of these bodies in nuclei of pericytes has never been previously reported. The origin and functional meaning of such structures is discussed in the light of recent ultrastructural and biochemical studies on nuclear inclusions.     

Metabolic responses of microbiota to diesel fuel addition in vegetated soil

The effects of trees and contamination on microbial metabolic activity, especially that of hydrocarbon degrading bacteria, were compared during phytoremediation to find which conditions increase diesel fuel removal. Diesel fuel utilisation, microbial extracellular enzyme activities and utilisation of Biolog ECO plate carbon sources by soil bacteria were determined during phytoremediation experiments consisting of two separate diesel applications. Diesel fuel removal after 28 days of second diesel application was 20–30% more than after the first application 1 year earlier. Soil microbiota utilised 26–31 of the 31 Biolog ECO plate carbon sources. Carbon source utilisation profiles indicated minor differences in microbiota in soil vegetated with pine compared to microbiota in soil vegetated with poplar. The potential maximum rates of aminopeptidase activity were 10–10² M AMC/h/g dry soil prior to and after second diesel application, except 14days after the second diesel addition, where the rates were at the scale of 10³M AMC/h/g dry soil. The potential maximum rates of esterase activity were 10³–10⁴M MUF/h/g dry soil. The presence of plants did not influence the activity of esterases. The utilisation of diesel by soil bacteria in Biolog MT2 plate assay was higher in contaminated soil, especially when vegetated, than in uncontaminated soil, measured both as lag times and maximum specific utilisation rates. MT2 plate assay detected the biological response after diesel fuel addition better than general activity methods.     

Visual edge fixation and negative phototaxis in the mealworm beetle Tenebrio molitor

The visual fixation response of the mealworm beetle Tenebrio molitor, elicited by black stripes upon a bright background is studied in an arena and by means of the Y-maze technique. In the arena the distribution n() of the beetle's angular position is measured at different distances from the centre, which is also the starting point. If the black stripe is narrow, the maximum of n() coincides with the centre of the stripe (centre-fixation Figure 1a). If one half of the panorama is black, the distribution n() has two maxima, which are near the borders between the black and white regions (edge-fixation Figure 1b). In the Y-maze experiments the beetle is tethered, but its head is free to move. The black stripes elicit turning tendencies F(), the strength of which depends upon the angular distance between the centre of the stripe and the animal's body axis. If the black stripe is narrow, the stable zero crossing of F() lies at =0, in agreement with the centre fixation in the arena (Fig. 3). If the stripe is 180° wide, two stable zero crossings are obtained near the border lines between the black and white regions, provided that the panorama is rotated around the animal with an angular velocity w larger than about 0.08°/s (Fig. 4). Below this value of w only one stable zero crossing at =0 exists (Fig. 6). Thus the tethered beetle's response underlies a transition between centre resp. edge fixation at a critical angular velocity of the drum. Some implications of this surprising phenomenon with respect to the mechanism of fixation and negative phototaxis are discussed but at present it is considered primarily a challenge for further investigation.Supported in part by Deutsche Forschungsgemeinschaft     

Total numbers of neurons in myenteric ganglia of the guinea-pig small intestine

Two techniques that are thought to stain all of the neurons in the myenteric ganglia of the intestine are NADH diaphorase histochemistry and immunhistochemistry using a nerve cell body antiserum. However, this assumption has never been directly verified. In the present study myenteric ganglia of the guinea-pig ileum were prepared as whole-mounts and stained with either of these techniques. All nerve cells that could be identified in the whole-mounts were counted. The whole-mounts were then embedded flat in resin and serially sectioned at 1 m. Nerve cells were identified and counted from the serial sections, and the data compared to those obtained from the whole-mounts. NADH diaphorase histochemistry did not reveal all the neurons at incubation times that gave selective staining. In contrast, nerve cell body antiserum stained the entire neuronal population. To determine the total number of nerve cell bodies/ganglion and the proportion of nerve cell bodies with calbindin immunoreactivity, whole-mounts that had been processed for calbindin immunohistochemistry were serially sectioned and reconstructed. The total number of neurons per myenteric ganglion was 105±10 (SE). Calbindin-immunoreactive neurons comprised about 20% of the myenteric neurons, which is considerably less than previous estimates, because previously the total population has been underestimated. The spatial density of myenteric neurons in the undistended ileum of the guinea-pig is 17300 nerve cells/cm².     

Chemoenzymatic galactosialylation with integrated cofactor regeneration

As a precursor for the chemical synthesis of sialylated oligosaccharides, the trisaccharide glycoside Neu5Ac (2-8)Gal(1-4)GlcNAc(1-O)-pent-4-ene was synthesized starting from GlcNAc(1-O)-pent-4-ene, UDP-glucose andN-acetylneuraminic acid in a one pot reaction employing galactosyltransferase and (2-6)sialyl-transferase in a complete cofactor regeneration system.Abbreviations Neu5Ac N-acetylneuraminic acid - CMP-Neu5Ac cytidine 5-monophosphosialate - CMP cytidine 5-monophosphate - CDP cytidine 5-diphosphate - CTP cytidine 5-triphosphate - Gal galactose - GlcNAc N-acetylglucosamine - UDP uridine 5-diphosphate - UDP-Glc uridine-5-diphosphoglucose - UDP-Gal uridine-5-diphosphogalactose - PEP phosphoenolpyruvate     

Ultrastructure and distribution of somatostatin-like immunoreactive neurons and nerve fibres in the coeliac ganglion of cats

Summary Somatostatin-like immunoreactivity was localized in nerve cell bodies and nerve terminals in the cat coeliac ganglion. Two types of somatostatin-immunoreactive cell bodies were revealed, the first being large (diameter 35 m), numerous and weakly labelled, where—as the second was considerably smaller (diameter 10.4 m), sparsely distributed and heavily stained. The immunoreactive nerve terminals were in synaptic contact with many immunonegative large neurons and dendrites. However, in a few cases, somatostatin-immunoreactive nerve terminals could also be observed on the surface of lightly stained neurons. Transection of vagal or mesenteric nerve failed to affect the distribution or density of somatostatin-like immunoreactive nerve terminals. These results demonstrate the existence of a synaptic input to the principal neurons of the coeliac ganglion of the cat by somatostatin-containing nerve terminals and suggest that this peptide may act as a neuromodulator or neurotransmitter. It is proposed that somatostatin-positive neurons provide intrinsic projections to other somatostatin-positive and to somatostatin-negative neurons throughout the coeliac ganglion, thereby creating a complex interneuronal system.     

Relations between Computational and Experimental Engineering Approaches to Molecules from Molecular Fragments

Computational techniques have become important tools in the preliminary stages of the design of new molecules. The mutual arrangements of interacting molecular parts and the required accessibility of important reactive groups on the peripheral regions of possible new molecules can be tested by computational means before the expensive and often complex synthetic methods are used to the actual construction of these molecules. There are some common features involved in the computational representation of molecular fragments and the synthetic methodologies used in the process of incorporating actual molecular fragments in such engineered molecules. One trend that appears to link these two approaches is based on the following observation: the greater local autonomy is shown by the calculated electron density contribution of a given molecular fragment, the more likely that this fragment can be regarded as a suitable building block of the molecule, and it is also more likely that there are convenient synthetic methods for the delivery of this fragment to its desired target location in the new molecule to be synthesized. For a precise formulation of this statement, a new concept, the degree of molecular fragment autonomy is introduced, using the inherent properties of molecular electron densities.